Among proteases, alkaline proteases to be incorporated into detergents such as laundry detergents are produced in the greatest amounts in the industrial field. Examples of such alkaline proteases known heretofore include Alcalase (registered trademark; Novozymes), Savinase (registered trademark; Novozymes), Maxacal (registered trademark; Genencor), Blap (registered trademark; Henkel), and KAP (Kao Corporation).
Protease is incorporated into a laundry detergent for providing the detergent with the ability to degrade protein soils deposited on clothing. However, actual soils deposited on clothing are complex soils containing, in addition to proteins, a plurality of organic and inorganic components such as sebum-derived lipid, mud, and dust. Therefore, demand has arisen for a detergent exhibiting excellent detergency against such complex soils.
In view of the foregoing, the present inventors have previously found several alkaline proteases having a molecular weight of about 43,000 which maintain sufficient casein-degrading activity even in the presence of fatty acids of high concentrations and which exhibit excellent detergency against complex soils containing proteins and sebum and have previously filed a patent application on the alkaline proteases (see International Publication WO99/18218). These alkaline proteases differ from conventionally known subtilisin, a serine protease derived from bacteria belonging to the genus Bacillus, in molecular weight, primary structure, and enzymological characteristics, particularly in having very strong oxidant resistance. Thus, it has been suggested that these alkaline proteases be classified into a new subtilisin subfamily (see Saeki, et al., Biochem. Biophys. Res. Commun., 279, (2000), 313–319).
However, the production amount of such proteases may be insufficient for industrial-scale production, and thus demand has arisen for an alkaline protease which is efficiently produced in a culture medium.
Meanwhile, in order to produce a large amount of the target protein (enzyme), attempts have been made to improve host bacteria (host strains) through mutation breeding, or to modify the gene encoding the enzyme or the gene controlling the expression of the enzyme, thereby enhancing secretion of the enzyme. However, no attempt has been made to enhance secretion of subtilisin by modifying the prepro sequence thereof, which plays an important role in generation and folding.